The present invention relates to a method of determining the importance of reducing oxidative stress in a specific hyperglycemic patient, and further to a method of determining a potential of a hyperglycemic patient to develop vascular complications in response to oxidative stress. More particularly, the present invention relates to determining a haptoglobin phenotype of a hyperglycemic patient as a means of predicting the likelihood of the patient developing vascular complications related to oxidative stress. More particularly, the present invention further relates to determining a haptoglobin phenotype of a hyperglycemic patient as a means of evaluating the potential efficacy of anti-oxidant therapy as an adjunct to treatment for a hyperglycemic patient.
Haptoglobin (Hp) is a hemoglobin-binding serum protein which plays a major role in the protection against heme-driven oxidative stress (Langlois M R and Delanghe J R (1996) Clin Chem 42: 1589-1600; Delanghe J R et al. (1998) AIDS 12: 1027-1032; Gutteridge J M. (1987) Biochim Biophys Acta 917: 219-223; Miller Y I et al. (1997) Biochem 36: 12189-12198; Vercellotti G M et al. (1994) Art Cell Blood Substit Imm Biotech 22: 207-213). Mice lacking the Hp gene demonstrate a dramatic increase in oxidative stress and oxidative tissue damage particularly in the kidney (Lim S K et al., (1998) Blood 92: 1870-1877).
In man, there are two common alleles for Hp (1 and 2) manifesting as three major phenotypes 1-1, 2-1 and 2-2 (Bowman B H and Kurosky A. (1982) Adv Hum Gen 12: 189-26).
Functional differences in the hemoglobin-binding capacity of the three phenotypes have been demonstrated. Hp in patients with the Hp 1-1 phenotype is able to bind more hemoglobin on per gram basis than Hps containing products of the Hp 2 allele (Langlois M R and Delanghe J R (1996) Clin Chem 42: 1589-1600). Hp molecules in patients with the Hp 1-1 phenotype are also more efficient antioxidants, since the smaller size of Hp 1-1 facilitates its entry to extravascular sites of oxidative tissue injury compared to products of the Hp 2 allele. This also includes a significantly greater glomerular sieving of Hp in patients with Hp 1-1 (Bowman B H and Kurosky A. (1982) Adv Hum Gen 12: 189-26). Differences in protection against oxidative stress in patients with the different Hp phenotypes results in differing serum levels of other antioxidants such as vitamin C (Langlois M R et al., (1997) Am J Clin Nutr 66: 606-610).
Long-term microvascular and macrovascular complications cause major morbidity and mortality in patients with diabetes mellitus (DM) (Diabetes Control and Complications Trial Research Group. (1993) N Eng J Med 329: 977-986). Four such complications are diabetic retinopathy (DR), diabetic nephropathy (DN), myocardial infarction and markedly increased risk of restenosis after percutaneous transluminal coronary angioplasty (PTCA). Approximately one third of patients with DM will develop end stage renal disease necessitating renal replacement therapy, within 25 years of the onset of diabetes. Epidemiological studies have demonstrated the important contributions of age of onset, duration, type of DM, and adequacy of metabolic control to the development and severity of DN (Diabetes Control and Complications Trial Research Group. (1993) N Eng J Med 329: 977-986.; Reichard P et al, (1993) N Eng J Med 329: 304-309). Patients with DM also manifest a 50 % increase in the rate of restenosis after PTCA (Carrozza J P et al. (1993) Ann Int Med 118: 344-349) compared to non-diabetics with an incidence as high as 70 % in some studies. This is particularly problematic in view of the increased incidence and extent of coronary artery disease in this population, often necessitating simultaneous treatment of multiple lesions in the same patient.
While numerous clinical trials have demonstrated anatomic predictors of restenosis in the diabetic and general populations (lesion length, vessel diameter) (Popma J J and Topol E J (1990) Am J Med 88: 16N-24N) there is currently little clinical information available to the clinician to predict which patients with DM are at greater risk for restenosis (Lincoff A M and Topol E J. (1997) Interventional catheterization techniques. In: Braunwald E, ed. Heart disease. 5.sup.th ed. Philadelphia: W B Saunders pp. 1372-1374).
DR is one of the four major causes of blindness in the US (Ferris F L. et al. (1999) N Engl J Med 341: 667-678). Epidemiological studies have demonstrated that the age of diabetes onset, the duration of the diabetes, and the adequacy of metabolic control influence the development and severity of DR. However, it is well recognized that not all patients with DM will develop DR and that not all patients with DR progress to sight-threatening proliferative DR.
Considerable evidence has demonstrated the importance of the generation of reactive oxygen species (oxidative stress) in the development of diabetic vascular complications (Low PA et al. (1997) Diabetes 46: S38-S42; Hotta N. (1997) Nagoya J Med Sc 60: 89-100; Khechai F. et al. (1997) Art Thromb Vasc Bio 17: 2885-2290; Vlassara H. et al. (1986) Clin Chem 32: B37-41; Dominguez C. et al. (1998) Diab Care 21: 1736-1742; Ceriello A. et al. (1996) Diab 45: 471-477; Guigliano D. et al.(1996) Diab Care 19: 257-267; Asayama K. et al. (1993) Free Rad Biol Med 15: 597-602; Pfeiffer A and Schatz H.(1995) Exp Clin End Diab 103: 7-14).
Persistent hyperglycemia results in glucose auto-oxidation, protein glycation products, increased prostanoid synthesis and protein kinase activation, all of which lead to the increased production of oxygen free radicals. Advanced glycation end product (AGE) (Makita Z., et al. (1991) N Eng J Med 325: 836-842) accumulation has been directly implicated in tissue damage associated with DN (Clements R S. et al. (1998) J Diab Comp 12: 28-33). AGE and related oxidation specific adducts, such as carboxymethylysine and malondialdehyde-lysine, have been demonstrated to accumulate in the mesangial matrix and nodular lesions of DN (Suzuki D. et al. (1999) J Am Soc Neph 10: 822-832; Suzuki D. and Miyata T. (1999) Int Med 38: 309-314; Horrie K. et al. (1997) J Clin Invest 100: 2995-3004). Levels of endogenous antioxidants such as vitamin C (Retsky K L. And Frei B. (1995) Biochim Biophys A 1257: 279-287) are decreased in patients with diabetic nephropathy, further augmenting the burden of oxidative stress (Hirsch I B. et al. (1998) J Diab Comp 12: 259-263). One potential mechanism of the benefit of AGE inhibitors on the development of DN (Lewis E J. et al. (1993) N Engl J Med 329: 1456-1462; Bain R. et al. (1992) J Am Soc Nephr 3: S97-103) may be their ability to reduce oxidative stress and the production and deposition of AGE in the diabetic kidney.
Multiple studies have demonstrated the importance of oxidative stress in restenosis after PTCA. Free hemoglobin released as a result of red blood cell fragmentation at the site of vascular injury (Jacob H S. (1994) J Lab Clin Med 123: 808-816) can act as a potent oxidizing agent. A change in the oxidation state of the vascular milieu at the site of balloon injury has been directly linked to apoptosis of medial smooth muscle cells (Pollman M J. et al. (1999) Circ Res; 84: 113-121), activation of cytomegalovirus (Speir E. et al. (1996) Circ Res 79: 1143-1152) and activation of transcription factors (Hofmann M A. et al. (1998) Diab Care 21: 1310-1316) such as NF-.kappa.B which mediate the inflammatory response to balloon injury. Antioxidant therapy has been demonstrated to have a beneficial effect on restenosis after PTCA (Rodes J. et al. (1998) Circulation 97: 429-436; Nunes G L. et al. (1995) Arterioscl Thromb Vasc Biol 15: 156-165; Schneider J E. et al. (1993) Circ 88: 628-637; Tardif J C. et al. (1997) New Engl J Med 337: 365-372).
Myocardial ischemia in diabetic patients is often more severe than in non diabetic patients due to the diffuse nature of the disease. In addition, diabetic patients have been shown to have fewer coronary artery collateral blood vessels that may serve to bypass coronary artery stenoses and serve as alternative conduits for blood flow. Currently there is no way to predict which patients are most likely to be affected by this problem. This is because collateral formation is highly variable between patients. This variability can only partially be explained by differences in the rate of the development of the coronary artery occlusive disease. Factors associated with a decreased collateral formation in animals and in man include diabetes, aging, hypercholesterolemia, hypertension and cigarette smoking. A genetic basis for collateral formation in the setting of coronary diseases has been described but remains controversial because no specific gene associated with collateral formation has been identified.
PCT WO98/37419 teaches a method and kit for determining a haptoglobin phenotype and specifically relates to applications involving human haptoglobin. Teachings of this patent focus on use of the haptoglobin 2-2 phenotype as an independent risk factor, specifically in relation to target organ damage in refractory essential hypertension, in relation to atherosclerosis (in the general population) and acute myocardial infarction and in relation to mortality from HIV infection. This patent does not teach the use of haptoglobin phenotype as a risk factor in vascular complications in DM. Because of the tendency of a haptoglobin 2-2 phenotype to make patients more prone to oxidative stress, it might be argued that use of a 2-2 phenotype as a negative predictor for vascular complications in DM is indirectly implied by this patent. However, teachings of this patent do not include the idea that haptoglobin 1-1 phenotype is a positive predictor for reduced tendency towards vascular complications in DM. Teachings of PCT WO98/37419 include use of a haptoglobin binding partner.
In other words, it is known that oxidative stress originating from Hp 2-1 or 2-2 phenotype leads to vascular complications in the general populations. It is also known that certain vascular complications are associated with oxidative stress associated with DM. It is, therefore plausible to assume the oxidative stress originating from either Hp 2-1 or 2-2 phenotype combined with that originating from DM will result in diabetes associated vascular complications. At present, it is, however, not known and cannot be predicted whether Hp1-1 phenotype mitigates the vascular complications in diabetic patients. This is the case, because DM and Hp1-1 phenotypes have opposing effects on the level of oxidative stress.
The binding partner according to PCT WO98/37419 may be any molecule with at least two locations by which it binds haptoglobin. The locations may be formed by a peptide, antibody, or a portion thereof, or by a lectin, a cell receptor, a molecular imprint or a bacterial antigen or a portion thereof. Teachings of this patent focus specifically on the use of the T4 antigen of S. pyogenes. All haptoglobins contain both alpha chains and beta chains. Beta chains are identical in all haptoglobins, while alpha chains differ between the two alleles of the haptoglobin gene. The alpha 2 chain of haptoglobin is the result of a mutation based on an unequal crossing over and includes 142 amino acids, in contrast to the 83 amino acids of the alpha 1 chain. Immunologically the alpha 1 and alpha 2 chains are similar, with the exception of a unique sequence of amino is acid residues in the alpha 2 chain (Ala-Val-Gly-Asp-Lys-Leu-Pro-Glu-Cys-Glu-Ala-Asp-Asp-Gly-Gln-Pro-Pro-Pro-L ys-Cys-Ile, SEQ ID NO:1). Any portion of this unique peptide sequence is therefore a suitable epitope for raising antibodies to differentiate between haptoglobins containing alpha 1 and alpha 2 chains as described in "Using Antibodies: A Laboratory Manual" (Ed Harlow and David Lane eds., Cold Spring Harbor Laboratory Press (1999)) which is fully incorporated herein by reference. Such antibodies might be monoclonal, polyclonal, or any portion thereof and may be enriched or purified by any one of a number of techniques known to those skilled in the art. In addition, the nucleotide sequence encoding this sequence can be readily employed to differentiate among Hp genotypes.
There is thus a widely recognized need for, and it would be highly advantageous to have a method to predict which specific DM patients have lower risk with respect to vascular complications. Such a method would allow medical practitioners to make best use of available resources while minimizing risk to each patient to the greatest possible extent.